Therapy for hepatitis C virus (HCV) infection has advanced rapidly with the recent approval of several direct-acting antivirals. However, most of the DAAs in clinical use or clinical trials target the same stage of HCV replication cycle and are associated with rapid emergence of drug-resistant viral mutations. In addition, different HCV genotypes and clinical conditions may also require adjustment of treatment regimen. Therefore, there is still an ongoing need to develop new HCV inhibitors that target different stages of the HCV replication cycle, such as entry and assembly. We developed a quantitative high-throughput screen (qHTS) assay platform with a cell-based HCV infection system. The highly sensitive assay can be miniaturized to 1536-well format for screening of large-scale chemical libraries. we performed a large-scale quantitative high-throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of about 350,000 chemicals and a library of approved pharmaceutical collection of about 3,000 compounds (the NPC library) for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial 3,000 molecule showing inhibitory activity for further structural analyses and functional assays. We were able to assign the majority of these compounds to specific stage(s) in the HCV replication cycle. These small molecules represent a diversity of chemotypes that are potential drug-like lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. From the NPC library, we identified chlorcyclizine HCl (CCZ), an over-the-counter drug for allergy symptoms, and related compounds as potent inhibitors of HCV infection. In an effort to optimize the CCZ class of compounds, we started a chemical/structural modification campaign, centered around chlorcyclazine, which resulted in optimized, non-chiral, nontoxic CCZ analogues with improved anti-HCV potency and pharmacokinetics that provide good coverage in liver at very reasonable doses. Lead compounds inhibited HCVsc infection without affecting HCVpp entry or HCV replication in the replicon assay, which is similar to that of CCZ, suggesting unaltered mechanism of action. Following our hit-to-lead optimization campaign, here we report an evaluation of preclinical in vitro ADME and in vivo pharmacokinetic profiles of several lead compounds, which led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. In the above HTS of the MLSMR library, we also identified an aryloxazole-based anti-HCV hit. Structure-activity relationship studies revealed several compounds exhibiting EC50 values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies showed high liver distribution and long half-life of these lead compounds without obvious hepatotoxicity. We further characterized compound 7ii and its mechanism of action, in vitro efficacy against various HCV genotypes, synergy with other FDA-approved HCV drugs, in vivo pharmacokinetics, and efficacy in a humanized mouse model against HCV genotype 1b, 2a and 3 infections. Timed addition of the compound in cell-based infection and membrane fusion assays indicate that this compound likely targets the viral fusion step. Cmpd 7ii showed antiviral synergy with FDA-approved HCV drugs in vitro, and it effectively inhibited all chimeric HCV genotypes although with varying EC50 values. From the mouse, rat, and canine pharmacokinetic studies, camped 7ii showed preferential localization in the liver with long T1/2 values (20 h) for PO and IV routes in both mice and rats. In the infected human hepatocyte-engrafted mice, camped 7ii monotherapy showed a more than 2-fold reduction in viral titer without clear emergence of drug resistance during a 4-week treatment for all HCV genotypes. In combination with daclatasvir (NS5A inhibitor), camped 7ii led to a sustained virologic response after 4 weeks of treatment. In summary, camped 7ii is a promising candidate in the next generation of combination drug cocktails for HCV treatment.